Isolation of hyodesoxycholic acid



Patented Apr. 3, 1951 2,547,726 ISOLATION OF HYODESOXYCHOLIC ACID ElwoodB. Trickey, Claymont, DeL, assignor to United States Rubber Company, NewYork,

N. Y., a corporation of New Jersey N Drawing. Application March 25,1949,

Serial No. 83,525

8 Claims. (Cl. 260397.1)

. bile by partial salting out of the sodium salt of alphaglycohyodesoxycholic acid with sodium chloride or other solubleinorganic salt, followed by caustic hydrolysis of the amide linkage inthe conjugated acid so obtained. The unconjugated acid obtained byacidification of the alkaline hydrolysis mixture may be filtered,

dried and recrystallized from ethyl acetate or other solvent. Hydrolysisof the amide link with NaOH or KOI-I gives the unconjugated acid(hyodesoxycholic acid) asthe alkali metal salt and also the alkali metalsalt of glycine. Acidificaticn of the strongly basic hydrolysis mixturewith hydrochloric acid releases the free hyodesoxycholic acid. Theglycine remains in solution as the hydrochloride.

This process, essentially that of Gumlich as described in Wieland etal., Zeitschrift fiir Physiologische Chemie 215, 18 (1933), requiresfresh hog bile and it is often difficult to produce pure hyodesoxycholicacid by this method. The yields of crude crystalline or pure crystallineproduct are also only 0.84 to 1.09% of the weight of the starting hobile.

The principal object of the present invention is to provide animprovedprocess of recovering hyodesoxycholic acid from hog bile whichgives much higher yields than have been obtainable heretofore. Anotherobject is to provide such a process which is applicable equally to freshor aged hog bile. Another object is to provide a process which rendersunnecessary a salting out procedure. Another object is to provide animproved method of crystallizing the hyodesoXycholic acid by effectingremoval of keto acids prior to the final crystallizing step. Numerousother objects Will more fully hereinafter appear.

I have found that if Whole hog bile is subjected to alkaline hydrolysisin the conventional way, which may be that described in the referencecited above, and that if this treatment is followed by a mineral acidhydrolysis, preferably in the presence of a solvent for the crude bileacids and preferably at refluxing temperature,

that a marked improvement in yield of hyode soxycholic acid results. Ihave found that a further improvement is effected by removing theketohydroxy acids present in the crude bile acid mixture, prior to finalcrystallization of the hyodesoxycholic acid, by means of a suitablereagent, for instance that of Anchel and Schoenheimer, J. Biol. Chem.124, 609-11 (1938) or of Ido and Sakurai, J. Biochem. (Japan) 29, 51-55(1939). Anchel et al. show the use of ketone reagents, typified byp-carboxyphenylhydrazine, for the separation of ketohydroxy acids.Typically the removal of these ketonic acids is effected by esterifyingthe crude acid acid mixture, treating with the ketone reagent anddistributing the reaction product between ether and dilute aqueouspotassium carbonate. The ketonic' acid esters couple with the ketonereagent and form water-soluble potassium salts which are dissolved inthe water phase. The hyodesoxycholic acid is recovered byrecrystallization of the crude acids derived from the separated etherphase.

I have found the removal of the keto acids in the foregoing mannergreatly aids the final crystallization of hyodesoxycholic acid and thusgives a substantial further improvement in yield thereof.

The acid hydrolysis step of my invention is carried out by heating thecrude bile acid mixture, resulting from the alkaline hydrolysis of thehog bile, with a mineral acid, preferably in aqueous solution, to asuitable elevated temperature. Any mineral acid, for example, phosphoricacid, sulfuric acid or hydrochloric acid, may be used. The selection ofa suitable amount and concentration of acid will be within the skill ofworkers in this art. The acid should be used in such strength and amountand the temperature and duration of heating should be such as to achievea substantial degree of hydrolysis. without causing any undesireddegradation. Typically, I use an amount of acid (measured as 100% acid)ranging from 30 to by weight of the crude bile acids resulting from thealkaline hydrolysis, and an acid concentration ranging from 10 to 25% byweight of the acid based on water and acid. The temperature almostinvariably will be within the range of C. to C. I prefer to carry outthe acid hydrolysis by refluxing the mixture. The duration of refluxingmay be several hours, say from 2 to 10 hours.

I prefer to conduct the acid hydrolysis under such conditions that thecrude bile acids remain in solution. This is achieved by having present'such an amount of a solvent capable of dissolving the crude bile acidsthat they remain in solution throughout this step. Examples of suitablesolvents are: water, aliphatic saturated alcohols, especially the loweralcohols especially those ranging from ethyl to amyl, ketones such asacetone and methyl ethyl ketone, etc. I find it especially desirable touse the lower alkanols such as those ranging from ethyl to amyl sincethese are very good solvents for the bile acids. When such loweralkanols are used, they esterify a part of the bile acids during thehydrolysis step. To avoid loss of bile acids as a result of this partialesterification, I prefer to subject the mixture resulting from acidhydrolysis in the presence of such lower alkanols to a further alkalinehydrolysis, by heating the mixture with alkali metal hydroxide tosaponify the esters thus formed. Thereafter the saponified mixture iscooled and acidified to precipitate the crude mixture of bile acidswhich is then treated in any suitable way to recover its hyodesoxycholicacid content in purified condition. The final purification of thehyodesoxycholic acid is preferably effected by crystallization from anysuitable organic solvent for hyodesoxycholic acid, ethyl acetate andacetone being especially suitable.

If an inert solvent such as water or a ketone is present during the acidhydrolysis, the mixture resulting from the acid hydrolysis may betreated in any suitable way to recover the hyodesoxycholic acid content,for example, by concentration by evaporation of solvent followed byrecovery of pure hyodesoxycholic acid from the precipitated crude bileacid mixture.

It is unnecessary to give details of the alkaline hydrolysis whichprecedes the acid hydrolysis, since it is merely the conventionalalkaline hydrolysis of hog bile which is well known, being described indetail in Gumlich, in Anchel et al., and in numerous other references.Typically, whole bile is heated, for example, boiled or autoclaved, with100% NaOH in an amount ranging from 5 to 20% by weight based on theweight of the whole bile for a period of time of from to 30 hours,cooled, diluted with water and neutralized with dilute I-ICl toprecipitate the crude bile acids which are then subjected to acidhydrolysis as detailed above.

Th reason for the improvement in yield due to the acid hydrolysis of theprocess of my invention is obscure but may be due to hydrolysis ofglycoside or other linkages amenable to attack with hot mineral acid andwhose presence would interfere with crystallization of thehyodesoxycholic acid. Using these improvements I have found that freshhog bile is not required, nor is a salting out procedure necessary, andthat the yields of hyodesoxycholic acid are 1.76% of th weight of thestarting bile, which is double the yield or nearly so, of that of theusually used procedure of Gumlich before referred to.

This discovery is new and surprising and makes readily available anabundant supply of a compound useful as a therapeutic agent or as anintermediate in the preparation of therapeutic agents.

The following examples serve to illustrate my invention.

EXAMPLE 1 Acid hydrolysis 183 grs. of the crude bile acids prepared byalkaline hydrolysis of whole hog bile according to the method describedby Anchel at al., cited above, are mixed with 1500 cc. of n-amylalcohol, 300 cc. of ethyl alcohol and 500 cc. of 18% 1-101 and refluxed3 hours. I believe that by this process any glycosidic or other linkagesamenable to attack with hot mineral acid are severed. At the same time aportion of the bile acids is esterified as a result of being heated inan alcohol in the presence of the mineral acid. Therefore to regain thefree acids the esters formed are saponified by another treatment withsodium hydroxide. After heating the acid mixture for 3 hours, as much aspossible of the uncombined alcohol is steam distilled off and enoughsodium hydroxide added to hydrolyze the esters formed and the solutionagain steam distilled to remove alcohol freed by hydrolysis. Thestrongly caustic solution is then cooled to 20 C. and acidified withdilute hydrochloric acid with stirring when a finely divided, nongummymixture of acids separates and is filtered, washed and dried at C. Theproduct resinifies upon drying at this temperature but can becrystallized from ethyl acetate or other solvent to give purehyodesoxycholic acid.

EXAMPLE 2 Removal of keto acids If the precipitated crudenon-crystalline mixture of bile acids obtained above beforecrystallizing from a solvent, is treated with a reagent to remove theketo hydroxy acids present as described for example by Anchel et al.,above cited, or by Ido et al., above cited, then a further improvementin yield can be effected.

Comparison of yields by old and present processes 150 grs. of the hogbile acids obtained by the procedure of Example 2, when dried and groundto a powder and boiled with 200 cc. of ethyl acetate, on coolingdeposits 41 grs. of crystalline hyodesoxycholic acid which melts at Onerecrystallization from acetone gives material melting at 196 C. Purehyodesoxycholic acid melts at 197 C. Gumlich gives as his yield 680 to880 grs. of hyodesoxycholic acid from 81 litres of hog bile. From anequivalent amount of bile by my process I can isolate 1425 grs.

Having thus described my invention, what I claim and desire to protectby Letters Patent is:

1. The process of obtaining hyodesoxycholic acid from hog bile whichcomprises subjecting the hog bile to an alkaline hydrolysis, thensubjecting the resulting bile materials to an acid hydrolysis by heatingwith a mineral acid, and recovering hyodesoxycholic acid from theresulting bile material.

2. The process of claim 1 wherein said acid hydrolysis is effected byheating with aqueous hydrochloric acid under refluxing conditions.

3. The process of claim 1 wherein said acid hydrolysis is conducted inthe presence of such an amount of a solvent for the bile acids that theyremain in solution throughout the acid hydrolysis.

4. The process of claim 1 wherein the crude bile acid mixture resultingfrom the acid hydrolysis is treated to remove the ketohydroxy acidspresent therein after which hyodesoxycholic acid is recovered from theresidual bile acid mixture by crystallization .from an organic solventfor hyodesoxycholic acid.

5. The process of claim 1 wherein said acid hydrolysis is effected byrefluxing a mixture of the crude bile acids resulting from the alkalinehydrolysis, aqueous hydrochloric acid and a lower saturated monohydricalcohol, and wherein the esters thus formed are thereafter saponified byheating with alkali metal hydroxide, the mixture is then acidified withprecipitation of a crude mixture of bile acids, and pure hyodesoxycholicacid is recovered from said mixture by crystallization from an organicsolvent for hyodesoxycholic acid.

6. The process of claim 5 wherein the crude mixture of bile acidsobtained upon acidification is treated to remove the ketohydroxy acidspresent before said crystallization step whereby a still furtherimprovement in yield of hyodesoxycholic acid is effected.

7. The process of obtaining hyodesoxycholic acid from hog bile whichcomprises heating the hog bile with alkali metal hydroxide to effectalkaline hydrolysis thereof, neutralizing the resulting mixture toprecipitate the crude bile acids, subjecting said crude bile acids to anacid hydrolysis by heating for from 2 to hours at from 90 C. to 125 C.in the presence of a 10 to aqueous solution of a mineral acid, theamount of said mineral acid, measured as 100% acid, ranging from to byweight of said crude bile acids, and recovering hyodesoxycholic acidfrom the resulting mixture.

8. The process of obtaining hyodesoxycholic acid from hog bile whichcomprises heating the hog bile with alkali metal hydroxide to effectalkaline hydrolysis thereof, neutralizing the resulting mixture toprecipitate the crude bile acids, subjecting said crude bile acids to anacid hydrolysis by heating for from 2 to 10 hours at from C. to 125 C.in the presence of a 10 to 25% aqueous solution of a mineral acid, theamount of said mineral acid, measured as% acid, ranging from 30 to 80%by weight of said crude bile acids and in the presence of a lowersaturated monohydric alcohol in such amount that the crude bile acidsremain in solution throughout the acid hydrolysis step, subjecting theresulting mixture to a further alkaline hydrolysis by heating withalkali metal hydroxide and thereby effecting saponification of esters ofthe bi1e acids and said alcohol formed during the acid hydrolysis,acidifying the resulting mixture to precipitate the crude mixture ofbile acids, and recovering hyodesoxycholic acid from said crude mixture.

ELWOOD B. TRICKEY.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Number Name Date 1,861,319 Peyer May 31, 19322,438,232 Sifierd Mar. 23, 1948

1. THE PROCESS OF OBTAINING HYODESOXYCHOLIC ACID FROM THE HOG BILE WHICHCOMPRISES SUBJECTING THE HOG BILE TO AN ALKALINE HYDROLYSIS, THENSUBJECTING THE RESULTING BILE MATERIALS TO AN ACID HYDROLYSIS BY HEATINGWITH A MINERAL ACID, AND RECOVERING HYODESOXYCHOLIC ACID FROM THERESULTING BILE MATERIAL.